Serine active site acetylcholinesterase

FIGURE 5.46 Interaction of the serine hydroxyl residue in the catalytically active site of acetylcholinesterase enzyme with esters of organophosphates or carbamates. The interaction leads to binding of the chemical with the enzyme, inhibition of the enzyme, inhibition of acetylcholine hydrolysis, and thus accumulation of acetylcholine in the synapses. 

This process of aging is believed to be critical in the development of delayed neuropathy, after NTE has been phosphorylated by an OP (see Chapter 10, Section 10.2.4). It is believed that most, if not all, of the B-esterases are sensitive to inhibition by OPs because they, too, have reactive serine at their active sites. It is important to emphasize that the interaction shown in Fignre 2.11 occurs with OPs that contain an oxon group. Phosphorothionates, which contain instead a thion group, do not readily interact in this way. Many OP insecticides are phosphorothionates, but these need to be converted to phosphate (oxon) forms by oxidative desulfuration before inhibition of acetylcholinesterase can proceed to any significant extent (see Section 2.3.2.2). 

Organophosphorus esters are known to react with a serine hydroxyl group in the active site of the acetylcholinesterase protein (Ecobichon 1991 Murphy 1986). Some organophosphorus esters (e.g., diisopropyl fluorophosphate, [DFP]) bind irreversibly, while others bind in a slowly reversible fashion, thereby leading to a slow reactivation (dephosphorylation) of the enzyme. A process known as "aging" has also been described in which reversibly bound compounds are changed with time to moieties that are essentially irreversibly... 

Most enzymes bind their substrates in a non-covalent manner but, for those that do bind covalently, the intermediate must be less stable than either substrate or product. Many of the enzymes that involve covalent catalysis are hydrolytic enzymes these include proteases, lipases, phosphatases and also acetylcholinesterase. Some of these enzymes possess a serine residue in the active site, which reacts with the substrate to form an acylenzyme intermediate that is attacked by water to complete the hydrolysis (Fignre 3.3). 

However, not included in the above mechanisms are other amino acid side-chains at the active site, whose special role will be to help bind the reagents in the required conformation for the reaction to occur. Examples of such interactions are found with acetylcholinesterase and chymotrypsin, representatives of a group of hydrolytic enzymes termed serine hydrolases, in that a specific serine amino acid residue is crucial for the mechanism of action. 

As a result, the penicillin occupies the active site of the enzyme, and becomes bound via the active-site serine residue. This binding causes irreversible enzyme inhibition, and stops cell-wall biosynthesis. Growing cells are killed due to rupture of the cell membrane and loss of cellular contents. The binding reaction between penicillinbinding proteins and penicillins is chemically analogous to the action of P-lactamases (see Boxes 7.20 and 13.5) however, in the latter case, penicilloic acid is subsequently released from the P-lactamase, and the enzyme can continue to function. Inhibitors of acetylcholinesterase (see Box 7.26) also bind irreversibly to the enzyme through a serine hydroxyl. 

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... 

Organophosphates form stable phosphoesters with the active site serine of acetylcholinesterase, the enzyme responsible for hydrolysis and inactivation of acetylcholine at cholinergic synapses. 

The answer is D. Organophosphates react with the active site serine residue of hydrolases such as acetylcholinesterase and form a stable phosphoester modification of that serine that inactivates the enzyme toward substrate. Inhibition of acetylcholinesterase causes overstimulation of the end organs regulated by those nerves. The symptoms manifested by this patient reflect such neurologic effects resulting from the inhalation or skin absorption of the pesticide diazinon. 

Carbamate Insecticides. Carbamate insecticides interact with acetylcholinesterase in exactly the same way as OPs, with the hydroxyl group in the serine at the enzyme s active site attacking the carbamate residue in the insecticide. However, the binding to the active site is reversible. Typical carbamate insecticides are shown in Figure 3.4. 

All of these esterases appear to act by mechanisms closely related to those of proteases. Acetylcholinesterase contains an active site serine that reacts with organophosphorus compounds (Box 12-E) and is part of an Asp-His-Ser catalytic triad which lies in a deep "gorge" as well as an oxyanion hole.637 A surprise is the absence of an essential carboxylate group that might bind the positively charged trimethylammonium... 

In the acylation step a nucleophilic group on one of the amino-acid side chains at the active site behaves as the nucleophile. As we have seen in Section 25-9B, the nucleophile of carboxypeptidase is the free carboxyl group of glutamic acid 270. In several other enzymes (chymotrypsin, subtilisin, trypsin, elastase, thrombin, acetylcholinesterase), it is the hydroxyl group of a serine residue ... 

A reaction looked at earlier simulates borate inhibition of serine proteinases.33 Resorufin acetate (234) is proposed as an attractive substrate to use with chymotrypsin since the absorbance of the product is several times more intense than that formed when the more usual p-nitrophcnyl acetate is used as a substrate. The steady-state values are the same for the two substrates, which is expected if the slow deacylation step involves a common intermediate. Experiments show that the acetate can bind to chymotrypsin other than at the active site.210 Brownian dynamics simulations of the encounter kinetics between the active site of an acetylcholinesterase and a charged substrate together with ah initio quantum chemical calculations using the 3-21G set to probe the transformation of the Michaelis complex into a covalently bound tetrahedral intermediate have been carried out.211 The Glu 199 residue located near the enzyme active triad boosts acetylcholinesterase activity by increasing the encounter rate due to the favourable modification of the electric field inside the enzyme and by stabilization of the TS for the first chemical step of catalysis.211... 

It is also important to mention the use of the reactivation of the acetylcholinesterase by pyridine-2-aldoxime methochloride to discriminate between the toxin and potential insecticides [96]. Once phos-phorylated, the active site serine of the enzyme can be reactivated by powerful nucleophilic agents such as oximes. However, this reactivation is not possible if attempted too late due to the stable adduct formed by the dealkylation (aging) of the inhibitor s remaining group. When acetylcholinesterase is inhibited by anatoxin-a(s), it shows immediately the characteristics of an aged enzyme and cannot be reactivated. In this way, it is possible to distinguish between the inhibition caused by anatoxin-a(s) and the one provoked by other insecticides. 

The serine proteases act by forming and hydrolyzing an ester on a serine residue. This was initially established using the nerve gas diisopropyl fluorophosphate, which inactivates serine proteases as well as acetylcholinesterase. It is a very potent inhibitor (it essentially binds in a 1 1 stoichiometry and thus can be used to titrate the active sites) and is extremely toxic in even low amounts. Careful acid or enzymatic hydrolysis (see Section 9.3.6.) of the inactivated enzyme yielded O-phosphoserine, and the serine was identified as residue 195 in the sequence. Chy-motrypsin acts on the compound cinnamoylimidazole, producing an acyl intermediate called cinnamoyl-enzyme which hydrolyzes slowly. This fact was exploited in an active-site titration (see Section 9.2.5.). Cinnamoyl-CT features a spectrum similar to that of the model compound O-cinnamoylserine, on denaturation of the enzyme in urea the spectrum was identical to that of O-acetylserine. Serine proteases act on both esters and amides. 

Mechanism of action Isoflurophate [eye soe FLURE oh fate] (diisopropylfluorophosphate, DFP) is an organophosphate that covalently binds to a serine-OH at the active site of acetylcholinesterase (Figure 4.9). Once this occurs, the enzyme is permanently inactivated, and restoration of acetylcholinesterase activity requires the synthesis of new enzyme molecules. Following covalent modification of acetylcholinesterase, the phosphorylated enzyme slowly releases one of its isopropyl groups (Figure 4.9). The loss of an alkyl group, which is called... 

Neurotransmitters are removed by translocation into vesicles or destroyed in enzyme-catalysed reactions. Acetylcholine must be removed from the synaptic cleft to permit repolarization and relaxation. A high affinity acetylcholinesterase (AChE) (the true or specific AChE) catalyses the hydrolysis of acetylcholine to acetate and choline. A plasma AChE (pseudo-AChE or non-specific AChE) also hydrolyses acetylcholine. A variety of plant-derived substances inhibit AChE and there is considerable interest in AChE inhibitors as potential therapies for cognition enhancement and for Alzheimer s disease. Organophosphorous compounds alkylate an active site serine on AChE and the AChE inhibition by this mechanism is the basis for the use of such compounds as insecticides (and unfortunately also as chemical warfare agents). Other synthetics with insecticidal and medical applications carbamoylate and thus inactivate AChE (Table 6.4). 

It is well known that organophosphates, carbamates and sulfonates are acid-transfer-inhibitors of serine hydrolases because they transfer the acid moiety of the inhibitor to the serine hydroxyl of the enzyme active site (34). Extensive evidence indicates that the reaction of these inhibitors with acetylcholinesterases (AChE) appears to involve the same reaction pathway as that for the esters of carboxylic acids, i.e. acetylcholine (see (35) for review), and in fact these inhibitors are considered to be poor substrates of AChE (36), especially the carbamic acid esters ("Equation 30 ). 

Acetylcholinesterase is only cholinesterase in insects. It is mainly located in the neuropile (area of synapses between nerve fibers) of the CNS in insects (Toutant, 1989). AChE contains two active sites, the esteratic site and the anionic site. The esteratic site possesses the hydroxyl group of serine and a basic nucleophilic imidazole group of histidine. The anionic site has a free carboxyl group (aspartic acid and/or glutamic acid). The interaction of ACh with AChE can be divided into three steps, as shown in Figure 7.13. The first... 

FIGURE 57.9. Inhibition and aging of serine esterases by diisopropylphosphorofluoridate (DFP). The active site serine is organophosphorylated in the inhibition step. Aging results in net loss of an isopropyl group to yield the monoisopropylphosphoryl esterase. This mode of inhibition and aging has been established for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and neuropathy target esterase catalytic domain (NEST) (Kropp and Richardson, 2007). 

Doom, J.A., Talley, T.T., Thompson, C.M., Richardson, R.J. (2001a). Probing the active sites of butyrylcholinesterase and cholesterol esterase with isomalathion conserved stereoselective inactivation of serine hydrolases stmcturally related to acetylcholinesterase. Chem. Res. Toxicol. 14 807-13. 

Nerve agents phosphorylate or phosphonylate the serine hydroxyl group at the esteratic part of the active site of the enzyme acetylcholinesterase (AChE EC 3.1.1.7) (Figure 66.2). AChE plays a key role in cholinergic transmission in the peripheral and central nervous system and, consequently, its inhibition is hfe-endangering (Taylor, 1996 Marrs, 1993). 

Serine residues occurring in the active sites of serine proteases (e.g., chymotryp-sin) or other hydrolyases (e.g., acetylcholinesterase) are specifically modified by diisopropylfluorophosphate in a reaction that is essentially irreversible. 

Most irreversible enzyme inhibitors combine covalently with functional groups at the active sites of enzymes. These inhibitors are usually chemically reactive, and many of them show some specificity in terms of the amino acid groups which they react with. Diisopropyl fluorophosphate (DFP), for example, forms a covalent adduct with active site serine residues, such as in the serine proteases, and in acetylcholinesterase, which explains its toxic effect on animals. Irreversible enzyme inhibition can be used to identify important active site residues. A special case of irreversible enzyme inhibition is the effect of suicide inhibitors, which are generally chemically unreactive compounds that resemble the substrate of the target enzyme and bind at the active site. The process of enzyme turnover begins, but the inhibitor is so... 

In a discovery project that is reminiscent of the discovery of captopril, scientists at Takeda created a hypothetical structure for the active site of acetylcholinesterase, based on SAR from previous biochemical and medicinal chemical work (141). The model consisted of (in addition to the serine protease-like catalytic machinery) an anionic binding site separating two discrete hydrophobic binding sites. This model was then used to design inhibitors of the enzyme (reviewed i n ref. 142). One set of analogs examined were based on the N-((o-phthalimidylalkyl)-iV-(a)-phenylalkyl)-amine (scaffold 66). An iterative process of testing. 

Figure 5. Variation of the protein MEP along the active sites of some enzymes. It a-chymotiypsin, 2t p-tiypsin, 3 porcine pancreatic elastase, 4 Streptomyces Griseus hydrolase, Si a-lytic protease, 6t subtilisin NOVO, 7i acetylcholinesterase, 8> lipase A, 9 lysozyme, lOi D-xyloie isomerase. Point A is at OG of die active serine in 1-8, at the bisector ofODl and OD2 of Asp-52 in 9, at Ol of the cyclic xylose m 10. Point B is atNE2 of the catalytic histidine in 1-8, in the first trisector of points A and Din 9, atNEl ofHis-S4in 10. Point C is at ND1 of the catalytic histidine in 1-8 and 10, at the second trisector of points A and D in 9. Point D is at the bisector of the carboxylate oxygens of the catalytic Asp or Glu side chains.

Acetylcholinesterase (AChE) catalyses the hydrolysis of the ester bond of acetylcholine to yield choline and acetate (Sussman et al., 1991). This is a critical reaction for the termination of impulses transmitted through cholinergic synapses. It is a highly efficient catalyst, with reaction rates approaching the diffusion limit. Its overall structure resembles the lipases with an active site gorge. Above the base of the gorge is the reactive serine to be activated by the classical (Ser-200...His-440...Glu-327) catalytic triad. 

While A-esterase(s) and B-esterases interact kinet-ically with paraoxon in a similar fashion (Figure 3), the molecular events occurring at their active sites during catalysis are probably very different. The active site of B-esterases such as acetylcholinesterase has been well characterized and contains a serine residue that is phosphorylated by paraoxon at the hydroxyl group. In contrast, the active site of A-est-erase(s) has not been studied as extensively, but it likely does not contain a serine residue that participates in the hydrolysis of paraoxon. Additionally, A-esterase(s) requires a divalent cation like calcium for activity, whereas B-esterases do not. 

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